EP0587799B1 - Inhibitors of cathepsin g and elastase for preventing connective tissue degradation - Google Patents

Inhibitors of cathepsin g and elastase for preventing connective tissue degradation Download PDF

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Publication number
EP0587799B1
EP0587799B1 EP92914209A EP92914209A EP0587799B1 EP 0587799 B1 EP0587799 B1 EP 0587799B1 EP 92914209 A EP92914209 A EP 92914209A EP 92914209 A EP92914209 A EP 92914209A EP 0587799 B1 EP0587799 B1 EP 0587799B1
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val
ala
formula
pro
group
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French (fr)
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EP0587799A4 (en
EP0587799A1 (en
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Michael R. Angelastro
Philippe Bey
Niall S. Doherty
Michael J. Janusz
Shujaath Mehdi
Norton P. Peet
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Aventis Pharmaceuticals Inc
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Merrell Dow Pharmaceuticals Inc
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/08—Tripeptides
    • C07K5/0827—Tripeptides containing heteroatoms different from O, S, or N
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/02—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing at least one abnormal peptide link
    • C07K5/021—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing at least one abnormal peptide link containing the structure -NH-(X)n-C(=0)-, n being 5 or 6; for n > 6, classification in C07K5/06 - C07K5/10, according to the moiety having normal peptide bonds
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/06—Dipeptides
    • C07K5/06191—Dipeptides containing heteroatoms different from O, S, or N
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/08—Tripeptides
    • C07K5/0802—Tripeptides with the first amino acid being neutral
    • C07K5/0804—Tripeptides with the first amino acid being neutral and aliphatic
    • C07K5/0806—Tripeptides with the first amino acid being neutral and aliphatic the side chain containing 0 or 1 carbon atoms, i.e. Gly, Ala
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/02—Linear peptides containing at least one abnormal peptide link
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • This invention relates to novel chemical compounds useful for preventing connective tissue degradation associated with neutrophil associated inflammatory disease.
  • Human neutrophil elastase and cathepsin G have been implicated in the tissue destruction associated with a number of inflammatory diseases such as chronic bronchitis, cystic fibrosis, and rheumatoid arthritis. H. L. Malech and J. I. Gallin, New Engl. J. Med., 317(11), 687 (1987). Both elastase and cathepsin G have a broad range of proteolytic activity against a number of connective tissue macromolecules including elastin, fibronectin, collagen, and proteoglycan. The presence of these enzymes may contribute to the pathology of these diseases.
  • European Patent Application No. 0410411 discloses inhibitors of various proteases, having a perfluoroethylketone moiety.
  • ⁇ -1-antiprotease ⁇ -1-PI
  • ⁇ -1-PI is a serine protease inhibitor that blocks the activity of both elastase and, at a slower rate, cathepsin G.
  • ⁇ -1-PI has received considerable interest because reduction in plasma levels to less than 15% of normal is associated with the early development of emphysema.
  • secretory fluids including bronchial, nasal, cervical mucus, and seminal fluid contain an endogenous protease inhibitor called secretory leukoprotease inhibitor (SLPI) that can inactivate both elastase and cathepsin G and is believed to play an important role in maintaining the integrity of the epithelium in the presence of inflammatory cell proteases.
  • SLPI secretory leukoprotease inhibitor
  • ⁇ -1-PI and SLPI are inactivated by neutrophil oxidative mechanisms allowing the neutrophil proteases to function in an essentially inhibitor-free environment.
  • ARDS adult respiratory distress syndrome
  • neutrophils possess non-oxidative mechanisms for eluding inhibition by antiproteases.
  • Neutrophils from patients with chronic granulomatous disease are capable of degrading endothelial cell matrices in the presence of excess ⁇ -1-PI.
  • stimulated neutrophils can tightly bind to their substrates such that serum antiproteases are effectively excluded from the microenviroment of tight cell-substrate contact.
  • the influx of large numbers of neutrophils to an inflammatory site may result in considerable tissue damage due to the proteolysis that occurs in this region.
  • elastase and cathepsin G are the primary neutrophil proteases responsible for cartilage matrix degradation as measured by the ability of neutrophil lysate, purified elastase and cathepsin G, and stimulated neutrophils to degrade cartilage matrix proteoglycan. Further, applicants have discovered that stimulated neutrophils degrade cartilage matrix in the presence of serum antiproteases indicating that degradation occurs in the serum-protected pericellular area between the neutrophils and substrate. Degradation of cartilage matrix occurring in the pericellular region could be blocked only by inhibiting both elastase and cathepsin G. Applicants have discovered a class of enzyme inhibitors which inhibit both elastase and cathepsin G and are thus useful in preventing neutrophil mediated connective tissue degradation.
  • Isosteres of the compounds of formula 1 include those wherein (a) one or more of the ⁇ -amino acid residues of the R 1 substituent is in its unnatural configuration (when there is a natural configuration) or (b) when the normal peptidic amide linkage is modified, such as for example, to form -CH 2 NH- (reduced), -COCH 2 - (keto), -CH(OH)CH 2 - (hydroxy), -CH(NH 2 )CH 2 - (amino), -CH 2 CH 2 - (hydrocarbon).
  • a compound of the invention should not be in an isosteric form; particularly it is preferred that there be no modified peptidic amide group in the R 1 group, but if there is, it is preferable to keep the isosteric modifications to a minimum.
  • the ⁇ -amino acid building blocks of these peptidase substrate analogs are preferably in their L-configuration.
  • the code for an amino acid wherein the first (or other) letter of the code is upper case indicates that the amino acid has the natural "L” configuration and wherein the first (or other) letter of the code is lower case indicates that the amino acid has "D” configuration.
  • Those compounds of this invention having aspartic or glutamic acid moieties may be in free form or a salt form, e.g., acid addition or anionic salt. Such a compound may be converted into its salt or base form in an art-known manner, one from another.
  • Preferred salts are trifluoroacetate, hydrochloride, sodium, potassium, or ammonium salts, although the scope of salts embraced herein is not limited thereto, the scope being extended to include all of the salts known to be used in the art of peptide chemistry.
  • Each ⁇ -amino acid has a characteristic "R-group", the R-group being the side chain, or residue, attached to the ⁇ -carbon atom of the ⁇ -amino acid.
  • R-group side chain for glycine is hydrogen, for alanine it is methyl, for valine it is isopropyl.
  • the R 2 moiety is the R-group for each indicated ⁇ -amino acid).
  • Those compounds of formula 1 wherein EIM or CGIM are a -C(O)C(O)R group can exist in a hydrated or unhydrated form. Hydrates of the triketo compounds having structure I' are much more chemically stable than are the unhydrated triketo compounds of formula 1 wherein EIM and/or CGIM is a -C(O)C(O)R group. For this reason, the hydrates are preferred and any reference in this specification and claims to a triketo compound should be taken to include reference to the corresponding hydrated form as context allows. Moreover, the compounds of this invention are expected to be in the hydrated form under normal physiological conditions.
  • CGIM and EIM is a -CF 3 or -CF 2 CF 3 group.
  • Applicants also especially prefer those compounds of formula 1 wherein -P 4 '-P 3 '-P 2 '-P 1 '- (SEQ ID NO: 4) is -Ala-Ala-Pro-Phe- (SEQ ID NO: 5); -Val-Pro-Phe-; or -Phe-.
  • SEQ ID NO: 4 is -Ala-Ala-Pro-Phe- (SEQ ID NO: 5); -Val-Pro-Phe-; or -Phe-.
  • the most preferred compound of this invention includes
  • the peptidase substrates of formula 1 are used for preventing connective tissue degradation such as cartilage degradation associated with neutrophil associated inflammatory disease and thus have an anti-inflammatory effect useful in the treatment of gout, rheumatoid arthritis and other inflammatory diseases, and to prevent elastin mediated tissue damage and thus can be used in the treatment of emphysema and adult respiratory disease syndrome (ARDS).
  • connective tissue degradation such as cartilage degradation associated with neutrophil associated inflammatory disease
  • ARDS adult respiratory disease syndrome
  • the enzyme inhibitory properties of the compounds of Formula 1 are readily ascertained by standard biochemical techniques well known in the art.
  • the preparation of the compounds of formula 1 may be achieved using standard chemical reactions analogously known to be useful for the preparation of a variety of known peptides.
  • the preferred manner of preparing the compounds of this invention is to first prepare fragments corresponding to the elastase inhibiting peptide of formula 2 and the cathepsin G inhibiting peptide cf formula 3, wherein P 1 , P 1 ', P 2 , P 2 ', P 3 , P 3 ', P 4 , P 4 ', EIM and CGIM are as defined for formula 1, and subsequently linking the two fragments with the "L" group.
  • the elastase inhibiting peptide of form 2 and the cathepsin G inhibiting peptide of form 3 are, in general, prepared by first preparing the compounds of structure 4 and structure 5, P 1 EIM P 1 '-CGIM wherein P 1 , P 1 ', EIM and CGIM are as defined for formula 1, or protected or activated derivatives thereof, and subsequently using standard techniques known to those skilled in the field of peptide chemistry to add the desired amino acids. For this purpose, a handy reference text for these techniques is the 1985 "The Practice of Peptide Synthesis" by M. Bodanszky and A.
  • Bodanszky wherein the parameters and techniques affecting the selection, use and removal of protective groups for individual and groups of amino acids is detailed, and which also contains activation and coupling techniques and other special procedures.
  • EIM elastase inhibiting moiety
  • CGIM cathepsin G inhibiting moiety
  • Those compounds of formulae 2 or 3 may be prepared using standard chemical reactions analogously known in the art. More specifically the compounds of formulae 2 and 3 wherein EIM and CGIM are -CF 2 H, -CF 3 , -CO 2 R 3 , -CONHR 3 , -C(O)R, -CF 2 CHR 3 C(O)NHR, H, alkyl, aryl, or aralkyl are known in art. Thus a description of the preparation of the compounds of formulae 2 or 3 wherein EIM or CGIM represents -CF 2 H, -CF 3 , -CO 2 R 3 , -CONHR 3 , or -C(O)R can be found in European Patent Application Number 195,212, published September 24, 1986.
  • the preparation of the compounds of formula 2 wherein EIM or CGIM represents -C(O)C(O)R is outlined in Scheme A wherein R, P 1 , P 2 , P 3 , and P 4 are as previously defined.
  • the compounds of formula 3 can be prepared in an analogous manner. Specifically, the compounds of formula 2 can be prepared by treatment of the appropriate ylide of formula 6 with (a) ozone and dimethyl sulfide or (b) singlet oxygen.
  • the ozonylysis reaction can be conveniently performed by, for example, bubbling an excess of ozone through a cooled solution of the appropriate formula 6 ylide.
  • Suitable solvents include any nonreactive solvent in which the formula 6 ylide is soluble, for example, alkyl esters of simple alkanoic acids such as ethyl acetate; the chlorinated hydrocarbons such as carbon tetrachloride, chloroform, 1,2-dichloroethane, 1,1,2,2-tetrachloroethane, and methylene chloride; the aromatic hydrocarbons such as benzene, toluene, and xylene; a chlorinated aromatic such as 1,2,4-trichlorobenzene and o -dichlorobenzene; an alcohol such as methanol, ethanol, and isopropanol; or an ethereal solvent such as diethyl ether, tetrahydrofuran (THF), and 1,4-dioxane.
  • Methylene chloride is preferred.
  • the temperature of the ozonolysis reaction mixture can be any temperature conducive to the reaction, typically from about -78°C to about 0°C, preferably from about -78°C to about -35°C, and most preferably about -70°C.
  • the time of the reaction will vary depending on the ylide, the concentration of the reactants, the temperature and other factors. Conveniently, ozone is bubbled into the reaction mixture until the solution turns blue indicating an excess of oxone.
  • the ozonide is then treated with an excess of a reducing agent such as zinc metal or preferably dimethylsulfide.
  • a reducing agent such as zinc metal or preferably dimethylsulfide.
  • the desired formula 2 compound as the hydrate is isolated from the reaction mixture in any convenient manner, typically by solvent removal (via evaporation). Purification may be accomplished by, for example, flash chromatography.
  • Oxidations utilizing singlet oxygen are well known. More specifically, singlet oxygen oxidation of an ylide to produce a tricarbonyl ester has been reported by H. Wasserman et al., J. Amer. Chem. Soc. 11 , 371 (1989).
  • Singlet oxygen can be generated by dye-sensitized excitation of oxygen.
  • Suitable dyes include Rose Bengal, Eosin Y and methylene blue.
  • Other sensitizers include dinaphthalenethiophene.
  • Rose Bengal and Eosin Y are attached to a basic anion-exchange resin and methylene blue is attached to an acidic cation-exchange resin. Excitation is accomplished with a UV lamp such as a tungsten-iodine lamp.
  • Suitable solvents are any solvents which promote and do not interfere with the desired reaction.
  • Such solvents include the aromatic hydrocarbons such as benzene and toluene; hydrocarbons such as hexane; ethereal solvents such as diethyl ether, tetrahydrofuran (THF), 1,4-dioxane; chlorinated hydrocarbons such as dichloromethane and chloroform; carbon disulfide; and alcohols such as methanol, ethanol, propanol, isopropanol and t-butanol. Mixtures are operable.
  • the temperature of the reaction mixture can be any suitable temperature from about -78°C to about 30°C typically from about -78°C to about -50°C.
  • the time of the reaction will vary depending on the reactant, the solvent, concentrations, and temperature and can be from about 1 min to about 2 hours. Purification and isolation can be by those methods described above for specification and isolation of product from the ozonolysis reaction mixture.
  • the formula 6 ylide is prepared from the appropriate N-protected ylide, preferably from the phthaloyl protected ylide of formula 7.
  • the removal of the phthaloyl group can be readily achieved by methods generally known to those skilled in the art.
  • a solution of the phthaloyl ylide can be allowed to react with hydrazine hydrate, typically about a 20-fold excess of hydrazine hydrate, until the reaction is substantially complete.
  • the solvent can be any of those described above for the ozonolysis reaction and preferably will be an alcohol solvent such as EtOH.
  • the temperature of the reaction mixture can be from about 0°C to about 60°C, conveniently at about room temperature, i.e., 25°C.
  • the reaction time will vary depending on the specific reactant, the temperature, the solvent, and other factors known to influence reaction time. Conveniently, the progress of the reaction can be monitored by thin layer chromatography (TLC).
  • the P 2 , P 3 , and P 4 groups can be linked to the now free amino group.
  • the P 2 , P 3 , and P 4 groups can be linked to the unprotected, free amino compound by well known peptide coupling techniques.
  • p -toluenesulfonyl (tosyl) moieties can be used to protect the amino side chains of amino acids such as Lys and Arg; p -methylbenzyl, acetamidomethyl, benzyl (Bzl), or t -butylsulfonyl moieties can be used to protect the sulfide containing side chains of amino acids such as cysteine, homocysteine, penicillamine and the like or derivatives thereof; benzyl (Bzl) or cyclohexyl ester moieties can be used to protect carboxylic acid side chains of amino acids such as Asp or Glu; a benzyl (Bzl) ether can be used to protect the hydroxy containing side chains of amino acids such as Ser and Thr; and a 2-bromocarbobenzoxy (Z(Br)) mo
  • side chain protecting groups are added and removed according to standard practices and procedures well known in the art. It is preferred to remove these side chain protecting groups with a solution of anisole in anhydrous hydrogen fluoride (1:10). Typically, removal of side chain protecting groups is performed after the peptide chain synthesis is complete but these groups can alternatively be removed at any other appropriate time. It is preferred to deprotect these side chains at the same time as the peptide is cleaved from the resin when solid phase synthetic methods are employed.
  • the phthaloyl ylide of formula 7 is prepared by reaction of the phthaloyl protected acid chloride of formula 8 with the phosphonium ylide of formula 9. This reaction is performed by adding a solution of the appropriate formula 9 ylide, preferably dropwise, to a solution of the formula 8 acid chloride.
  • Suitable solvents include those listed above for the ozonolysis reaction and will preferably be an ethereal solvent such as THF.
  • the reaction will require from about 30 minutes to about 12 hours, typically about 2 to 3 hours, depending on the acid chloride, the ylide, the solvent(s), and the temperature which can be from about 0°C to about 60°C, conveniently at about room temperature, i.e., 25°C.
  • Isolation and purification is accomplished by filtering the reaction mixture to remove solid products and subsequently chromatographing the filtrate, for example, on silica gel eluting with a 50% mixture of ethyl acetate and hexane.
  • the formula 9 phosphorous ylide, Wittig reagent is prepared from the corresponding formula 10 ⁇ -halocarboxylic acid derivative in the usual manner, that is, by reacting the ⁇ -halo ester with a tertiary phosphine such as triphenylphosphine to yield a phosphonium salt.
  • a strong base such as an organolithium compound, for example, lithium diisopropylamide (LDA), sodium hydride, or sodium amide, the acidic proton is removed and the desired ylide is formed.
  • LDA lithium diisopropylamide
  • sodium hydride sodium amide
  • Suitable solvents used in forming the Wittig reagent include any nonreactive solvent, for example, the aromatic hydrocarbons such as benzene or toluene, the chlorinated hydrocarbons such as carbon tetrachloride, chloroform, or methylene chloride, or the ethereal solvents such as diethyl ether or THF.
  • the aromatic hydrocarbons such as benzene or toluene
  • the chlorinated hydrocarbons such as carbon tetrachloride, chloroform, or methylene chloride
  • the ethereal solvents such as diethyl ether or THF.
  • the reaction can conveniently be performed at from about 0°C to about 60°C, typically at room temperature, that is about 25°C.
  • the halo group of the ⁇ -halo ester is preferably a bromo group, but can be a chloro or iodo group or can be any good leaving group which forms a stable phosphonium salt such as a mesylate or tosylate group.
  • the acid chloride of formula 8 is prepared from the corresponding acid of formula 11 by, for example, reacting the acid with refluxing ⁇ , ⁇ -dichloromethyl methylether. After about 3 hours, the solution is allowed to cool and the product concentrated by solvent evaporation. The resulting crude acid chloride can be used directly without further purification in the reaction with the formula 9 phosphorous ylide.
  • the compounds of this invention are prepared by reducing the N-methoxy-N-methyl amide of either formula 15a or 15b to produce the aldehydes of formulae 14a and 14b, respectively.
  • the reduction can be performed in any way generally known and readily performed by those skilled in the art such as by use of lithium aluminum hydride (LAH).
  • LAH lithium aluminum hydride
  • This reduction can be conveniently carried out by adding an excess of LAH to a cooled, typically about 0°C, solution of a formula 15a or 15b compound in a nonreactive solvent such as an ethereal solvent such as tetrahydrofuran (THF).
  • a nonreactive solvent such as an ethereal solvent such as tetrahydrofuran (THF).
  • reaction mixture is quenched by the addition of, for example, 10% potassium hydrogen sulfate and then water.
  • the product can then be isolated by, for example, extraction of the aqueous mixture with a solvent such as ethyl acetate, drying and solvent removal.
  • the crude product can be purified by, for example, column chromatography such as a silica gel column eluting with 55% ethyl acetate/hexane or recrystallization.
  • the formulae 14a and 14b aldehydes are then reacted with the pentafluoroethyl anion, such as the lithium salt of the pentafluoroethyl anion to give the alcohols of formulae 13a or 13b, respectively.
  • pentafluoroethyl anion such as the lithium salt of the pentafluoroethyl anion to give the alcohols of formulae 13a or 13b, respectively.
  • the perfluoroethyl anion is generated in situ by addition of methyllithium/lithium bromide complex to a solution of the aldehyde and pentafluoroethyl iodide in a nonreactive solvent such as diethyl ether.
  • a nonreactive solvent such as diethyl ether.
  • the cooled (-78° - 0°C) reaction mixture is allowed to stir for about one-half to about 1 hour or until the reaction is substantially complete and then the mixture is quenched by pouring into an excess of dilute hydrochloric acid.
  • the product is isolated by, for example, extraction with diethyl ether and subsequent solvent removal.
  • the crude product is purified by, for example, chromatography on silica gel.
  • the alcohols of formulae 13a or 13b are then oxidized to give the amino-protected pentafluoroethyl ketones of formula 12 or the desired product of formula 2, respectively.
  • the oxidation may be effected via the well-known Swern oxidation procedure, or with a modified Jones oxidation using pyridinium dichromate, or a chromic anhydride-pyridinium complex, or with the Dess-Martin periodinane, 1,1,1-tris(acetyloxy)-1,1-dihydro-1,2-benzoiodoxol-3(1H)-one.
  • the coupling procedures are effected according to standard procedures well known in the art.
  • the Swern oxidation is effected by reacting about 2 to 10 equivalents of dimethylsulfoxide (DMSO) with about 1 to 6 equivalents of trifluoroacetic anhydride [(CF 3 CO) 2 O] or oxalyl chloride [(COCl) 2 ], said reactants being dissolved in an inert solvent, e.g., methylene chloride (CH 2 Cl 2 ), said reaction being under an inert atmosphere (e.g., nitrogen or equivalently functioning gas) under anhydrous conditions at temperatures of about -80°C to -50°C to form an in situ sulfonium adduct to which is added about 1 equivalent of an appropriate alcohol of formula 13a or 13b.
  • DMSO dimethylsulfoxide
  • COCl oxalyl chloride
  • the alcohols are dissolved in an inert solvent, e.g., CH 2 Cl 2 or minimum amounts of DMSO, and the reaction mixture is allowed to warm to about -50°C (for about 10-20 minutes) and then the reaction is completed by adding about 3 to 10 equivalents of a tertiary amine, e.g., triethylamine, N-methylmorpholine, etc.
  • an inert solvent e.g., CH 2 Cl 2 or minimum amounts of DMSO
  • the modified Jones oxidation procedure may conveniently be effected by reacting an alcohol of formula 13a or 13b with pyridinium dichromate by contacting the reactants together in a water-trapping molecular sieve powder, (e.g., a powdered 3 Angstrom molecular sieve), wherein said contact is in the presence of glacial acetic acid at about 0°C to 50°C, preferably at room temperature followed by isolation and then optionally removing amine protecting groups.
  • a water-trapping molecular sieve powder e.g., a powdered 3 Angstrom molecular sieve
  • a chromic anhydride-pyridine complex i.e., a Sarett reagent prepared in situ (see Fieser and Fieser "Reagents for Organic Synthesis” Vol. 1, pp. 145 and Sarett, et al., J.A.C.S. 25 , 422, (1953)
  • said complex being prepared in situ in an inert solvent (e.g., CH 2 Cl 2 ) in an inert atmosphere under anhydrous conditions at 0°C to 50°C to which complex is added 1 equivalent of an alcohol of formula 13a or 13b allowing the reactants to interact for about 1 to 15 hours, followed by isolation and optionally removing amine protecting groups.
  • Another alternative process for converting an alcohol of formula 13a or 13b to the desired ketone of formula 1 or 5 is an oxidation reaction which employs Dess-Martin periodinane (see Dess and Martin, J. Org. Chem. , 48 , 4155, (1983)).
  • This oxidation is effected by contacting about 1 equivalent of the appropriate alcohol of formula 13a or 13b with 1 to 5 equivalents of periodinane (preferably 1.5 equivalents), said reagent being in suspension in an inert solvent (e.g., methylene chloride) under an inert atmosphere (preferably nitrogen) under anhydrous conditions at 0°C to 50°C (preferably room temperature) and allowing the reactants to interact for about 1 to 48 hours.
  • an inert solvent e.g., methylene chloride
  • an inert atmosphere preferably nitrogen
  • the formula compounds are prepared by first converting the amino-protected, perfluoroethyl alcohol of formula 13a to the corresponding compound of formula 13b, prior to final oxidation.
  • the amino-protected, perfluoroethyl alcohol of formula 13a is first deprotected, if desired, and then any amino acids or peptide chain represented by P 4 -P 3 -P 2 - can be added using standard ⁇ -amino acid or peptide coupling procedures.
  • the P 4 -P 3 -P 2 - group is made up of more than one amino acid
  • either the entire peptide chain can be added to the deprotected formula 13a compound or the amino acids can be coupled to the deprotected formula 13a compound sequentially. Alternatively, a combination of these two coupling methods can be used.
  • the compounds of formula 12 can be converted to the desired formula 2 compounds.
  • p -toluenesulfonyl (tosyl) moieties can be used to protect the amine side chains of amino acids such as Lys and Arg; p -methylbenzyl, acetamidomethyl, benzyl (Bzl), or t -butylsulfonyl moieties can be used to protect the sulfide containing side chains of amino acids such as cysteine, homocysteine, penicillamine and the like or derivatives thereof; benzyl (Bzl) or cyclohexyl ester moieties can be used to protect carboxylic acid side chains of amino acids such as Asp, Glu; a benzyl (Bzl) ether can be used to protect the hydroxy containing side chains of amino acids such as Ser and Thr; and a 2-bromocarbobenzoxy (Z(Br))
  • side chain protecting groups are added and removed according to standard practices and procedures well known in the art. It is preferred to remove these side chain protecting groups with a solution of anisole in anhydrous hydrogen fluoride (1:10). Typically, removal of side chain protecting groups is performed after the peptide chain synthesis is complete but these groups can alternatively be removed at any other appropriate time. It is preferred to deprotect these side chains at the same time as the peptide is cleaved from the resin when solid phase synthetic methods are employed.
  • the compounds of formulae 15a and 15b can be converted directly to the compounds of formulae 12 or 2, respectively, by condensation of the N-methoxy-N-methyl amide with the lithium salt of the perfluoroethyl anion in the same manner in which the compounds of formulae 15a and 15b are converted to the compounds of formulae 14a and 14b, respectively.
  • the compounds are then isolated and purified by standard techniques.
  • the desired amino acids, derivatives and isomers thereof can be obtained commercially or can be synthesized according to standard practices and procedures well known in the art.
  • N-methoxy-N-methyl amides of formulae 15a and 15b are prepared from the corresponding ⁇ -amino acids of formulae 16a and 16b, wherein R 1 and R 2 are as defined for formula 1 and wherein Pg is an amino protecting group such as carbamate, preferably a benzyloxycarbonyl (Cbz) group, respectively, in the usual manner.
  • Pg is an amino protecting group such as carbamate, preferably a benzyloxycarbonyl (Cbz) group, respectively, in the usual manner.
  • Isobutylchloroformate is added to a cooled (i.e.
  • N-methylmorpholine or another sterically hindered, non-nucleophilic tertiary amine and an ⁇ -amino acid compound in a nonrective solvent such as methylene chloride After about 5 minutes to about 1 hour, typically about 15 - 20 minutes, N,O-dimethylhydroxylamine HCl is added and the mixture allowed to stir for from about 30 minutes up to about 6 hours and then the reaction mixture is allowed to warm to room temperature. When the reaction is substantially complete, typically after about 1 to about 10 hours, the mixture is poured into water and the aqueous phase is extracted with, for example, ethyl acetate.
  • the desired compound is then isolated by solvent evaporation and crude purification can be accomplished by, for example, flash chromatography on silica gel eluting with ethyl acetate/hexane. Purification can be acomplished by, for example, flash chromatography on silica gel eluting with methylene chloride.
  • Scheme C provides a general synthetic procedure for preparing the compounds of formula 1 wherein L 1 and L 2 are both represented by carbonyl groups, L 1 and L 2 are both represented by sulfonyl groups or L 2 is represented by a carbonyl group and L 1 is represented by a sulfonyl group.
  • step a the appropriate elastase inhibiting peptide fragment of formula 2 is coupled with the appropriate derivative of L as described by structure (17) to give the corresponding L-elastase inhibiting peptide fragment of structure (18) by techniques well known in the art.
  • an appropriate derivative of L as described by structure (17) is one wherein the L 2 carbonyl group is represented by a t-butyloxycarbonyl protected carboxylic acid and the L 1 carbonyl group is represented by an unprotected carboxylic acid.
  • an appropriate derivative of L as described by structure (17) is one wherein the L 1 sulfonyl group is represented by a sulfonyl chloride group and the L 2 sulfonyl group is represented by an unprotected sulfonic acid.
  • an appropriate derivative of L as described by structure (17) is one wherein the L 2 carbonyl group is represented by a t-butyloxycarbonyl protected carboxylic acid and the L 1 sulfonyl group is represented by a sulfonyl chloride group.
  • step b the appropriate L-elastase inhibiting peptide fragment of structure (18) is coupled with the appropriate cathepsin G inhibiting peptide fragment of formula 3 to give the corresponding compound of formula 1 by techniques well known in the art.
  • the appropriate L-elastase inhibiting peptide fragment of structure (18) is one wherein the L 2 carbonyl group is represented by a t-butyloxycarbonyl protected carboxylic acid
  • the t-butyloxycarbonyl protected carboxylic acid must first be hydrolyzed by techniques well known in the art prior to the coupling reaction in step b.
  • Scheme D provides a general synthetic procedure for preparing the compounds of formula 1 wherein L 1 is represented by a carbonyl group and L 2 is represented by a sulfonyl group.
  • step a the appropriate cathepsin G inhibiting peptide fragment of formula 3 is coupled with the appropriate derivative of L as described by structure (19) to give the corresponding cathepsin G inhibiting peptide fragment of structure (20) by techniques well known in the art.
  • L 1 carbonyl group is represented by a t-butyloxycarbonyl protected carboxylic acid and the L 2 carbonyl group is represented by a sulfonyl chloride group.
  • step b the appropriate cathepsin G inhibiting peptide fragment of structure (20) is coupled with the appropriate elastase inhibiting peptide fragment of formula 2 to give the corresponding compound of formula 1 by techniques well known in the art.
  • Val-Pro-Val[CF 2 CF 3 ]•hydrochloride 200mg between ethyl acetate (10mL) and saturated sodium hydrogen carbonate (20mL). Separate the organic phase and extract the aqueous phase with ethyl acetate (3X20mL). Combine the organic phases, dry (MgSO 4 ) and evaporate the solvent in vacuo to give Val-Pro-Val[CF 2 CF 3 ].
  • Val-Pro-Phe[CF 3 ]•hydrochloride 200mg
  • ethyl acetate 10mL
  • saturated sodium hydrogen carbonate 20mL
  • ethyl acetate 3X20mL
  • Val-Pro-Val[CF 2 CF 3 ] (568mg, 1.37mmol) and Val-Pro-Phe[CF 3 ] (500mg, 1.37mmol) in methanol (20mL) and add glyoxal (200mg of a 40% solution in water, 1.37mmol), sodium cyanoborohydride (86mg, 1.37mmol) and 1 drop of 1% bromocresol green in ethanol. Maintain the pH of the reaction with 1N hydrochloric acid in methanol until the indicator no longer changes. Evaporate the solvent in vacuo and partition the residue between 1N sodium hydroxide (5mL) and ethyl acetate (10mL). Separate the organic phase, dry (MgSO 4 ) and evaporate the solvent invacuo. Purify by chromatography to give the title compound.

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Abstract

Novel compounds which are chemically linked inhibitors of the proteases Elastase and Cathepsin G prevent connective tissue degradation associated with neutrophil induced inflammatory disease.

Description

  • This invention relates to novel chemical compounds useful for preventing connective tissue degradation associated with neutrophil associated inflammatory disease.
  • Human neutrophil elastase and cathepsin G have been implicated in the tissue destruction associated with a number of inflammatory diseases such as chronic bronchitis, cystic fibrosis, and rheumatoid arthritis. H. L. Malech and J. I. Gallin, New Engl. J. Med., 317(11), 687 (1987). Both elastase and cathepsin G have a broad range of proteolytic activity against a number of connective tissue macromolecules including elastin, fibronectin, collagen, and proteoglycan. The presence of these enzymes may contribute to the pathology of these diseases.
  • European Patent Application No. 0410411 discloses inhibitors of various proteases, having a perfluoroethylketone moiety.
  • Normal plasma contains large quantities of protease inhibitors that control a variety of enzymes involved in connective tissue turnover and inflammation. For example, α-1-antiprotease (α-1-PI) is a serine protease inhibitor that blocks the activity of both elastase and, at a slower rate, cathepsin G. α-1-PI has received considerable interest because reduction in plasma levels to less than 15% of normal is associated with the early development of emphysema.
  • In addition to plasma derived protease inhibitors, secretory fluids, including bronchial, nasal, cervical mucus, and seminal fluid contain an endogenous protease inhibitor called secretory leukoprotease inhibitor (SLPI) that can inactivate both elastase and cathepsin G and is believed to play an important role in maintaining the integrity of the epithelium in the presence of inflammatory cell proteases. In certain pathological states α-1-PI and SLPI are inactivated by neutrophil oxidative mechanisms allowing the neutrophil proteases to function in an essentially inhibitor-free environment. For example, bronchial lavage fluids from patients with adult respiratory distress syndrome (ARDS) have been found to contain active elastase and α-1-PI that had been inactivated by oxidation.
  • In addition to oxidative mechanisms, neutrophils possess non-oxidative mechanisms for eluding inhibition by antiproteases. Neutrophils from patients with chronic granulomatous disease are capable of degrading endothelial cell matrices in the presence of excess α-1-PI. There is considerable in vitro evidence that stimulated neutrophils can tightly bind to their substrates such that serum antiproteases are effectively excluded from the microenviroment of tight cell-substrate contact. The influx of large numbers of neutrophils to an inflammatory site may result in considerable tissue damage due to the proteolysis that occurs in this region.
  • Applicants have determined that elastase and cathepsin G are the primary neutrophil proteases responsible for cartilage matrix degradation as measured by the ability of neutrophil lysate, purified elastase and cathepsin G, and stimulated neutrophils to degrade cartilage matrix proteoglycan. Further, applicants have discovered that stimulated neutrophils degrade cartilage matrix in the presence of serum antiproteases indicating that degradation occurs in the serum-protected pericellular area between the neutrophils and substrate. Degradation of cartilage matrix occurring in the pericellular region could be blocked only by inhibiting both elastase and cathepsin G. Applicants have discovered a class of enzyme inhibitors which inhibit both elastase and cathepsin G and are thus useful in preventing neutrophil mediated connective tissue degradation.
    • SUMMARY OF THE INVENTION
  • Compounds of the formula
    Figure 00030001
    wherein
    • P1
    is Ala, bAla, Leu, Ile, Val, Nva, bVal, Met, Nle, Gly, or Sar;
    P1'
    is Ala, bAla, Leu, Ile, Val, Nva, bVal, Met, Nle, Phe, Tyr, Tyr(Me), Ala(3pyr), Ala(4pyr), Trp, or Nal(l);
    P2
    is Pro, Ind, Ala, bAla, Leu, Ile, Val, Nva, bVal, Met, Nle, Phe, Tyr, Tyr(Me), Ala(3pyr), Ala(4pyr), Trp, or Nal(1);
    P2'
    is Pro, Ind, Ala, bAla, Leu, Ile, Val, Nva, bVal, Met, Nle, Gly, Sar or is absent;
    P3
    is Lys, Arg, Pro, Ind, Ala, bAla, Leu, Ile, Val, Nva, bVal, Met, or Nle;
    P3'
    is Ala, bAla, Leu, Ile, Val, Nva, bVal, Met, Nle, Gly, Sar or is absent;
    P4
    is Ala, bAla, Leu, Ile, Val, Nva, bVal, Met, Nle, or is absent;
    P4'
    is Ala, bAla, Leu, Ile, Val, Nva, bVal, Met, Nle, Gly, Sar, or is absent;
    L
    is a group of one of the formulae -C(O)-(CH2)n-C(O)- -C(O)-(CH2)p-(CH = CH)r-(CH2)q-C(O)- ― L1 ― Ph ― L2 ― ― L1 ― Ph1 ― B ― Ph2 ― L2 ― -(CH2)n+2- -C(O)- wherein
    • n is 0 or an integer of from 1 to 6;
    • p and q are each independently an integer of from 1 to 6;
    • r is 1 or 2;
    • L1 and L2 are each independently selected from a carbonyl or sulfonyl group wherein L1 is bound to the elastase inhibiting fragment and L2 is bound to the cathepsin G inhibiting fragment;
    • Ph, Ph1, and Ph2 are each independently a m-phenylene or p-phenylene group;
    • B is a bond, -(CH2)m-, or a -S(O)2N(H)C(O)- group;
    EIM and CGIM are each independently selected from the group consisting of -C(O)C(O)R, -CF2CF3, -CF3, -CF2H, CO2R3, -CONHR3, -CF2CHR3C(O)NHR, -H, alkyl, aryl, aralkyl, -C(O)R,
    wherein
    • R3 is H, alkyl, phenyl, benzyl,
    • R is OH or alkoxy
    or a pharmaceutically acceptable salt thereof are useful in the prevention of cartilage degradation. DETAILED DESCRIPTION OF THE INVENTION
  • Isosteres of the compounds of formula 1 include those wherein (a) one or more of the α-amino acid residues of the R1 substituent is in its unnatural configuration (when there is a natural configuration) or (b) when the normal peptidic amide linkage is modified, such as for example, to form -CH2NH- (reduced), -COCH2- (keto), -CH(OH)CH2- (hydroxy), -CH(NH2)CH2- (amino), -CH2CH2- (hydrocarbon). Preferably a compound of the invention should not be in an isosteric form; particularly it is preferred that there be no modified peptidic amide group in the R1 group, but if there is, it is preferable to keep the isosteric modifications to a minimum.
  • Unless otherwise stated, the α-amino acid building blocks of these peptidase substrate analogs are preferably in their L-configuration. As is conventional nomenclature used by peptide chemists, the code for an amino acid wherein the first (or other) letter of the code is upper case indicates that the amino acid has the natural "L" configuration and wherein the first (or other) letter of the code is lower case indicates that the amino acid has "D" configuration. Throughout this specification reference will be made to lower case amino acid codes or codes proceeded by "(D)-" and these shall both be taken as equivalent.
  • Those compounds of this invention having aspartic or glutamic acid moieties may be in free form or a salt form, e.g., acid addition or anionic salt. Such a compound may be converted into its salt or base form in an art-known manner, one from another. Preferred salts are trifluoroacetate, hydrochloride, sodium, potassium, or ammonium salts, although the scope of salts embraced herein is not limited thereto, the scope being extended to include all of the salts known to be used in the art of peptide chemistry.
  • Before further defining and/or illustrating the scope of the peptidase inhibitors embraced by formula 1, it may be convenient to state some of the more basic concepts related to peptides. Each α-amino acid has a characteristic "R-group", the R-group being the side chain, or residue, attached to the α-carbon atom of the α-amino acid. For example, the R-group side chain for glycine is hydrogen, for alanine it is methyl, for valine it is isopropyl. (Thus, throughout this specification the R2 moiety is the R-group for each indicated α-amino acid). For the specific R-groups - or side chains - of the α-amino acids reference to A.L. Lehninger's text on Biochemistry (see particularly Chapter 4) is helpful.
  • Those compounds of formula 1 wherein EIM or CGIM are a -C(O)C(O)R group can exist in a hydrated or unhydrated form. Hydrates of the triketo compounds having structure I' are much more chemically stable than are the unhydrated
    Figure 00070001
    triketo compounds of formula 1 wherein EIM and/or CGIM is a -C(O)C(O)R group. For this reason, the hydrates are preferred and any reference in this specification and claims to a triketo compound should be taken to include reference to the corresponding hydrated form as context allows. Moreover, the compounds of this invention are expected to be in the hydrated form under normal physiological conditions.
  • The recognized abbreviations for the α-amino acids are set forth in Table I
    AMINO ACID SYMBOL
    Alanine Ala
    Arginine Arg
    Aspargine Asn
    Aspartic acid Asp
    Asn + Asp Asx
    Cysteine Cys
    Glutamine Gln
    Glutamic acid Glu
    Gln + Glu Glx
    Glycine Gly
    Histidine His
    Isoleucine Ile
    Leucine Leu
    Lysine Lys
    Methionine Met
    Phenylalanine Phe
    p-Guanidinophenylalanine Phe(Gua)
    Proline Pro
    Serine Ser
    Threonine Thr
    Tryptophan Trp
    Tyrosine Tyr
    Valine Val
    Norvaline Nva
    Norleucine Nle
    1-Naphthylalanine Nal(1)
    2-Indolinecarboxylic acid Ind
    Sarcosine Sar
    Cyclohexylalanine Cha
    beta-Alanine bAla
    beta-Valine bVal
    O-4'-Methyltyrosine Tyr(Me)
    3-Pyrazolylalanine Ala(3pyr)
    4-Pyrimidinylalanine Ala(4pyr)
    N6-(2-carboxybenzoyl)lysine Lys(2CBz)
    Terephthalyl tPht
    N6-acetyllysine Lys(Ac)
  • Applicants prefer those compounds of formula 1 wherein P1 is norvaline or valine. Applicants also prefer those compounds of formula 1 wherein P1 is norvaline or valine; P1' is phenylalanine; P2 is proline; P2' is proline; P3 is isoleucine, valine, or alanine; P3' is alanine, valine, or is absent; P4 is alanine or is absent; and wherein P4' is alanine or is absent. Applicants prefer those compounds wherein L is a -C(O)-phenylene-C(O)- group, especially wherein the phenylene is a para-phenylene group. Applicants prefer those compounds of formula 1 wherein CGIM and EIM is a -CF3 or -CF2CF3 group. Applicants especially prefer those compounds of formula 1 wherein -P4-P3-P2-P1- (SEQ ID NO: 2) is -Ala-Ala-Pro-Val- (SEQ ID NO: 3); Lys(2CBz)-Pro-Val-; or -Val-Pro-Val- group. Applicants also especially prefer those compounds of formula 1 wherein -P4'-P3'-P2'-P1'- (SEQ ID NO: 4) is -Ala-Ala-Pro-Phe- (SEQ ID NO: 5); -Val-Pro-Phe-; or -Phe-. The most preferred compound of this invention includes
    Figure 00100001
  • The peptidase substrates of formula 1 are used for preventing connective tissue degradation such as cartilage degradation associated with neutrophil associated inflammatory disease and thus have an anti-inflammatory effect useful in the treatment of gout, rheumatoid arthritis and other inflammatory diseases, and to prevent elastin mediated tissue damage and thus can be used in the treatment of emphysema and adult respiratory disease syndrome (ARDS). In their end-use application the enzyme inhibitory properties of the compounds of Formula 1 are readily ascertained by standard biochemical techniques well known in the art. Potential dose range for their end-use application will of course depend upon the nature and severity of the disease state as determined by the attending diagnostician with the range of 0.01 to 10 mg/kg body weight per day being useful for the aforementioned disease states with 0.1 mg to 10 mg/kg per day being preferred.
  • Having defined the scope of compounds embraced within the generic formula 1, the manner in which such compounds may be prepared will herein below be described as illustrated. The preparation of the compounds of formula 1 may be achieved using standard chemical reactions analogously known to be useful for the preparation of a variety of known peptides. The preferred manner of preparing the compounds of this invention is to first prepare fragments corresponding to the elastase inhibiting peptide of formula 2 and the cathepsin G inhibiting peptide cf formula 3,
    Figure 00110001
    Figure 00110002
    wherein P1, P1', P2, P2', P3, P3', P4, P4', EIM and CGIM are as defined for formula 1, and subsequently linking the two fragments with the "L" group. The elastase inhibiting peptide of form 2 and the cathepsin G inhibiting peptide of form 3 are, in general, prepared by first preparing the compounds of structure 4 and structure 5, P1EIM P1'-CGIM wherein P1, P1', EIM and CGIM are as defined for formula 1, or protected or activated derivatives thereof, and subsequently using standard techniques known to those skilled in the field of peptide chemistry to add the desired amino acids. For this purpose, a handy reference text for these techniques is the 1985 "The Practice of Peptide Synthesis" by M. Bodanszky and A. Bodanszky, wherein the parameters and techniques affecting the selection, use and removal of protective groups for individual and groups of amino acids is detailed, and which also contains activation and coupling techniques and other special procedures. However, before the application of these peptide chemistry techniques may be applied, certain key intermediates containing the elastase inhibiting moiety (EIM) and the cathepsin G inhibiting moiety (CGIM) must first be prepared. The preparation of the key intermediates is described as follows.
  • Those compounds of formulae 2 or 3 may be prepared using standard chemical reactions analogously known in the art. More specifically the compounds of formulae 2 and 3 wherein EIM and CGIM are -CF2H, -CF3, -CO2R3, -CONHR3, -C(O)R, -CF2CHR3C(O)NHR, H, alkyl, aryl, or aralkyl are known in art. Thus a description of the preparation of the compounds of formulae 2 or 3 wherein EIM or CGIM represents -CF2H, -CF3, -CO2R3, -CONHR3, or -C(O)R can be found in European Patent Application Number 195,212, published September 24, 1986. A description of the preparation of the compounds of formulae 2 or 3 wherein EIM or CGIM represents -CF2CHR3C(O)NHR are described in European Application Number 275,101, published July 20, 1988. A description of the preparation of the compounds of formulae 2 and 3 wherein EIM and CGIM represents H, alkyl, aryl, or aralkyl are described in European Patent Application Number 363,284, published April 11, 1990.
  • The preparation of the compounds of formula 2 wherein EIM or CGIM represents -C(O)C(O)R is outlined in Scheme A wherein R, P1, P2, P3, and P4 are as previously defined. The compounds of formula 3 can be prepared in an analogous manner. Specifically, the compounds of formula 2 can be prepared by treatment of the appropriate ylide of formula 6 with (a) ozone and dimethyl sulfide or (b) singlet oxygen. The ozonylysis reaction can be conveniently performed by, for example, bubbling an excess of ozone through a cooled solution of the appropriate formula 6 ylide. Suitable solvents include any nonreactive solvent in which the formula 6 ylide is soluble, for example, alkyl esters of simple alkanoic acids such as ethyl acetate; the chlorinated hydrocarbons such as carbon tetrachloride, chloroform, 1,2-dichloroethane, 1,1,2,2-tetrachloroethane, and methylene chloride; the aromatic hydrocarbons such as benzene, toluene, and xylene; a chlorinated aromatic such as 1,2,4-trichlorobenzene and o-dichlorobenzene; an alcohol such as methanol, ethanol, and isopropanol; or an ethereal solvent such as diethyl ether, tetrahydrofuran (THF), and 1,4-dioxane. Methylene chloride is preferred.
    Figure 00140001
  • The temperature of the ozonolysis reaction mixture can be any temperature conducive to the reaction, typically from about -78°C to about 0°C, preferably from about -78°C to about -35°C, and most preferably about -70°C. The time of the reaction will vary depending on the ylide, the concentration of the reactants, the temperature and other factors. Conveniently, ozone is bubbled into the reaction mixture until the solution turns blue indicating an excess of oxone.
  • The ozonide is then treated with an excess of a reducing agent such as zinc metal or preferably dimethylsulfide. The desired formula 2 compound as the hydrate is isolated from the reaction mixture in any convenient manner, typically by solvent removal (via evaporation). Purification may be accomplished by, for example, flash chromatography.
  • Oxidations utilizing singlet oxygen are well known. More specifically, singlet oxygen oxidation of an ylide to produce a tricarbonyl ester has been reported by H. Wasserman et al., J. Amer. Chem. Soc. 11, 371 (1989).
  • Singlet oxygen can be generated by dye-sensitized excitation of oxygen. Suitable dyes include Rose Bengal, Eosin Y and methylene blue. Other sensitizers include dinaphthalenethiophene. Typically, Rose Bengal and Eosin Y are attached to a basic anion-exchange resin and methylene blue is attached to an acidic cation-exchange resin. Excitation is accomplished with a UV lamp such as a tungsten-iodine lamp. Suitable solvents are any solvents which promote and do not interfere with the desired reaction. Such solvents include the aromatic hydrocarbons such as benzene and toluene; hydrocarbons such as hexane; ethereal solvents such as diethyl ether, tetrahydrofuran (THF), 1,4-dioxane; chlorinated hydrocarbons such as dichloromethane and chloroform; carbon disulfide; and alcohols such as methanol, ethanol, propanol, isopropanol and t-butanol. Mixtures are operable. The temperature of the reaction mixture can be any suitable temperature from about -78°C to about 30°C typically from about -78°C to about -50°C. The time of the reaction will vary depending on the reactant, the solvent, concentrations, and temperature and can be from about 1 min to about 2 hours. Purification and isolation can be by those methods described above for specification and isolation of product from the ozonolysis reaction mixture.
  • The formula 6 ylide is prepared from the appropriate N-protected ylide, preferably from the phthaloyl protected ylide of formula 7. The removal of the phthaloyl group can be readily achieved by methods generally known to those skilled in the art. For example, a solution of the phthaloyl ylide can be allowed to react with hydrazine hydrate, typically about a 20-fold excess of hydrazine hydrate, until the reaction is substantially complete. The solvent can be any of those described above for the ozonolysis reaction and preferably will be an alcohol solvent such as EtOH. The temperature of the reaction mixture can be from about 0°C to about 60°C, conveniently at about room temperature, i.e., 25°C. The reaction time will vary depending on the specific reactant, the temperature, the solvent, and other factors known to influence reaction time. Conveniently, the progress of the reaction can be monitored by thin layer chromatography (TLC).
  • Subsequent to removal of the phthaloyl group, the P2, P3, and P4 groups can be linked to the now free amino group. The P2, P3, and P4 groups can be linked to the unprotected, free amino compound by well known peptide coupling techniques.
  • In coupling individual amino acids or peptides to the deprotected formula 7 compound, appropriate side chain protecting groups are employed. The selection and use of an appropriate protecting group for these side chain functionalities is within the ability of those skilled in the art and will depend upon the amino acid to be protected and the presence of other protected amino acid residues in the peptide. The selection of such a side chain protecting group is critical in that it must not be removed during the deprotection and coupling steps of the synthesis. For example, when Boc is used as the α-amino protecting group, the following side chain protecting groups are suitable: p-toluenesulfonyl (tosyl) moieties can be used to protect the amino side chains of amino acids such as Lys and Arg; p-methylbenzyl, acetamidomethyl, benzyl (Bzl), or t-butylsulfonyl moieties can be used to protect the sulfide containing side chains of amino acids such as cysteine, homocysteine, penicillamine and the like or derivatives thereof; benzyl (Bzl) or cyclohexyl ester moieties can be used to protect carboxylic acid side chains of amino acids such as Asp or Glu; a benzyl (Bzl) ether can be used to protect the hydroxy containing side chains of amino acids such as Ser and Thr; and a 2-bromocarbobenzoxy (Z(Br)) moiety can be used to protect the hydroxy containing side chains of amino acids such as Tyr. These side chain protecting groups are added and removed according to standard practices and procedures well known in the art. It is preferred to remove these side chain protecting groups with a solution of anisole in anhydrous hydrogen fluoride (1:10). Typically, removal of side chain protecting groups is performed after the peptide chain synthesis is complete but these groups can alternatively be removed at any other appropriate time. It is preferred to deprotect these side chains at the same time as the peptide is cleaved from the resin when solid phase synthetic methods are employed.
  • The phthaloyl ylide of formula 7 is prepared by reaction of the phthaloyl protected acid chloride of formula 8 with the phosphonium ylide of formula 9. This reaction is performed by adding a solution of the appropriate formula 9 ylide, preferably dropwise, to a solution of the formula 8 acid chloride. Suitable solvents include those listed above for the ozonolysis reaction and will preferably be an ethereal solvent such as THF. The reaction will require from about 30 minutes to about 12 hours, typically about 2 to 3 hours, depending on the acid chloride, the ylide, the solvent(s), and the temperature which can be from about 0°C to about 60°C, conveniently at about room temperature, i.e., 25°C. Isolation and purification is accomplished by filtering the reaction mixture to remove solid products and subsequently chromatographing the filtrate, for example, on silica gel eluting with a 50% mixture of ethyl acetate and hexane.
  • The formula 9 phosphorous ylide, Wittig reagent, is prepared from the corresponding formula 10 α-halocarboxylic acid derivative in the usual manner, that is, by reacting the α-halo ester with a tertiary phosphine such as triphenylphosphine to yield a phosphonium salt. When treated with a strong base such as an organolithium compound, for example, lithium diisopropylamide (LDA), sodium hydride, or sodium amide, the acidic proton is removed and the desired ylide is formed. Suitable solvents used in forming the Wittig reagent include any nonreactive solvent, for example, the aromatic hydrocarbons such as benzene or toluene, the chlorinated hydrocarbons such as carbon tetrachloride, chloroform, or methylene chloride, or the ethereal solvents such as diethyl ether or THF.
  • The reaction can conveniently be performed at from about 0°C to about 60°C, typically at room temperature, that is about 25°C. The halo group of the α-halo ester is preferably a bromo group, but can be a chloro or iodo group or can be any good leaving group which forms a stable phosphonium salt such as a mesylate or tosylate group.
  • The acid chloride of formula 8 is prepared from the corresponding acid of formula 11 by, for example, reacting the acid with refluxing α,α-dichloromethyl methylether. After about 3 hours, the solution is allowed to cool and the product concentrated by solvent evaporation. The resulting crude acid chloride can be used directly without further purification in the reaction with the formula 9 phosphorous ylide.
  • The preparation of the compounds of formula 2 wherein EIM or CGIM represents -CF2CF3 is outlined in Scheme B wherein R1 and R2 are as previously defined, and Pg is an amino protecting group such as a carbamate, preferably a benzyloxycarbonyl (Cbz) group.
    Figure 00200001
    The compounds of formula 3 can be prepared in an analogous manner.
  • Specifically the compounds of this invention are prepared by reducing the N-methoxy-N-methyl amide of either formula 15a or 15b to produce the aldehydes of formulae 14a and 14b, respectively. Applicants prefer to use the compounds of formula 15a as the initial starting materials. The reduction can be performed in any way generally known and readily performed by those skilled in the art such as by use of lithium aluminum hydride (LAH). This reduction can be conveniently carried out by adding an excess of LAH to a cooled, typically about 0°C, solution of a formula 15a or 15b compound in a nonreactive solvent such as an ethereal solvent such as tetrahydrofuran (THF). After the reaction is substantially complete, typically after about 30 minutes, the reaction mixture is quenched by the addition of, for example, 10% potassium hydrogen sulfate and then water. The product can then be isolated by, for example, extraction of the aqueous mixture with a solvent such as ethyl acetate, drying and solvent removal. The crude product can be purified by, for example, column chromatography such as a silica gel column eluting with 55% ethyl acetate/hexane or recrystallization.
  • The formulae 14a and 14b aldehydes are then reacted with the pentafluoroethyl anion, such as the lithium salt of the pentafluoroethyl anion to give the alcohols of formulae 13a or 13b, respectively. This condensation can be conveniently performed by those skilled in the art by a modified procedure as described by P. G. Gassman and Neil J. O'Reilly, J. Org. Chem. 1987, 52, 2481-2490. In this procedure, the perfluoroethyl anion is generated in situ by addition of methyllithium/lithium bromide complex to a solution of the aldehyde and pentafluoroethyl iodide in a nonreactive solvent such as diethyl ether. The cooled (-78° - 0°C) reaction mixture is allowed to stir for about one-half to about 1 hour or until the reaction is substantially complete and then the mixture is quenched by pouring into an excess of dilute hydrochloric acid. The product is isolated by, for example, extraction with diethyl ether and subsequent solvent removal. The crude product is purified by, for example, chromatography on silica gel.
  • The alcohols of formulae 13a or 13b are then oxidized to give the amino-protected pentafluoroethyl ketones of formula 12 or the desired product of formula 2, respectively. The oxidation may be effected via the well-known Swern oxidation procedure, or with a modified Jones oxidation using pyridinium dichromate, or a chromic anhydride-pyridinium complex, or with the Dess-Martin periodinane, 1,1,1-tris(acetyloxy)-1,1-dihydro-1,2-benzoiodoxol-3(1H)-one. Of course, if there are any protecting groups on the residues of the α-amino acid building blocks, such protecting groups may be removed after oxidation. The coupling procedures are effected according to standard procedures well known in the art.
  • In general the Swern oxidation is effected by reacting about 2 to 10 equivalents of dimethylsulfoxide (DMSO) with about 1 to 6 equivalents of trifluoroacetic anhydride [(CF3CO)2O] or oxalyl chloride [(COCl)2], said reactants being dissolved in an inert solvent, e.g., methylene chloride (CH2Cl2), said reaction being under an inert atmosphere (e.g., nitrogen or equivalently functioning gas) under anhydrous conditions at temperatures of about -80°C to -50°C to form an in situ sulfonium adduct to which is added about 1 equivalent of an appropriate alcohol of formula 13a or 13b. Preferably, the alcohols are dissolved in an inert solvent, e.g., CH2Cl2 or minimum amounts of DMSO, and the reaction mixture is allowed to warm to about -50°C (for about 10-20 minutes) and then the reaction is completed by adding about 3 to 10 equivalents of a tertiary amine, e.g., triethylamine, N-methylmorpholine, etc.
  • In general, the modified Jones oxidation procedure may conveniently be effected by reacting an alcohol of formula 13a or 13b with pyridinium dichromate by contacting the reactants together in a water-trapping molecular sieve powder, (e.g., a powdered 3 Angstrom molecular sieve), wherein said contact is in the presence of glacial acetic acid at about 0°C to 50°C, preferably at room temperature followed by isolation and then optionally removing amine protecting groups.
  • Alternatively, 1 to 5 equivalents of a chromic anhydride-pyridine complex (i.e., a Sarett reagent prepared in situ (see Fieser and Fieser "Reagents for Organic Synthesis" Vol. 1, pp. 145 and Sarett, et al., J.A.C.S. 25, 422, (1953)) said complex being prepared in situ in an inert solvent (e.g., CH2Cl2) in an inert atmosphere under anhydrous conditions at 0°C to 50°C to which complex is added 1 equivalent of an alcohol of formula 13a or 13b allowing the reactants to interact for about 1 to 15 hours, followed by isolation and optionally removing amine protecting groups.
  • Another alternative process for converting an alcohol of formula 13a or 13b to the desired ketone of formula 1 or 5 is an oxidation reaction which employs Dess-Martin periodinane (see Dess and Martin, J. Org. Chem., 48, 4155, (1983)). This oxidation is effected by contacting about 1 equivalent of the appropriate alcohol of formula 13a or 13b with 1 to 5 equivalents of periodinane (preferably 1.5 equivalents), said reagent being in suspension in an inert solvent (e.g., methylene chloride) under an inert atmosphere (preferably nitrogen) under anhydrous conditions at 0°C to 50°C (preferably room temperature) and allowing the reactants to interact for about 1 to 48 hours. Optional deprotection of the amine protecting groups may be effected as desired after the ketones have been isolated.
  • In one mode of preparing the compounds of this invention the formula compounds are prepared by first converting the amino-protected, perfluoroethyl alcohol of formula 13a to the corresponding compound of formula 13b, prior to final oxidation. The amino-protected, perfluoroethyl alcohol of formula 13a is first deprotected, if desired, and then any amino acids or peptide chain represented by P4-P3-P2- can be added using standard α-amino acid or peptide coupling procedures. Where the P4-P3-P2- group is made up of more than one amino acid, either the entire peptide chain can be added to the deprotected formula 13a compound or the amino acids can be coupled to the deprotected formula 13a compound sequentially. Alternatively, a combination of these two coupling methods can be used. In a like manner, the compounds of formula 12 can be converted to the desired formula 2 compounds.
  • In coupling individual amino acids or peptides to the deprotected formula 13a or formula 12 compounds, appropriate side chain protecting groups are employed. The selection and use of an appropriate protecting group for these side chain functionalities is within the ability of those skilled in the art and will depend upon the amino acid to be protected and the presence of other protected amino acid residues in the peptide. The selection of such a side chain protecting group is critical in that it must not be removed during the deprotection and coupling steps of the synthesis. For example, when Boc is used as the α-amino protecting group, the following side chain protecting groups are suitable: p-toluenesulfonyl (tosyl) moieties can be used to protect the amine side chains of amino acids such as Lys and Arg; p-methylbenzyl, acetamidomethyl, benzyl (Bzl), or t-butylsulfonyl moieties can be used to protect the sulfide containing side chains of amino acids such as cysteine, homocysteine, penicillamine and the like or derivatives thereof; benzyl (Bzl) or cyclohexyl ester moieties can be used to protect carboxylic acid side chains of amino acids such as Asp, Glu; a benzyl (Bzl) ether can be used to protect the hydroxy containing side chains of amino acids such as Ser and Thr; and a 2-bromocarbobenzoxy (Z(Br)) moiety can be used to protect the hydroxy containing side chains of amino acids such as Tyr. These side chain protecting groups are added and removed according to standard practices and procedures well known in the art. It is preferred to remove these side chain protecting groups with a solution of anisole in anhydrous hydrogen fluoride (1:10). Typically, removal of side chain protecting groups is performed after the peptide chain synthesis is complete but these groups can alternatively be removed at any other appropriate time. It is preferred to deprotect these side chains at the same time as the peptide is cleaved from the resin when solid phase synthetic methods are employed.
  • In the preferred mode of preparing the compounds of this invention, the compounds of formulae 15a and 15b can be converted directly to the compounds of formulae 12 or 2, respectively, by condensation of the N-methoxy-N-methyl amide with the lithium salt of the perfluoroethyl anion in the same manner in which the compounds of formulae 15a and 15b are converted to the compounds of formulae 14a and 14b, respectively.
  • The compounds are then isolated and purified by standard techniques. The desired amino acids, derivatives and isomers thereof can be obtained commercially or can be synthesized according to standard practices and procedures well known in the art.
  • The N-methoxy-N-methyl amides of formulae 15a and 15b are prepared from the corresponding α-amino acids of formulae 16a and 16b, wherein R1 and R2 are as defined for formula 1 and wherein Pg is an amino protecting group such as carbamate, preferably a benzyloxycarbonyl (Cbz) group, respectively, in the usual manner. (See, for example, J.A. Fehrentz and B. Castro, Synthesis, 676-78 (1983).
    Figure 00260001
    Isobutylchloroformate is added to a cooled (i.e. -60°C to about 0°C) mixture of N-methylmorpholine or another sterically hindered, non-nucleophilic tertiary amine and an α-amino acid compound in a nonrective solvent such as methylene chloride. After about 5 minutes to about 1 hour, typically about 15 - 20 minutes, N,O-dimethylhydroxylamine HCl is added and the mixture allowed to stir for from about 30 minutes up to about 6 hours and then the reaction mixture is allowed to warm to room temperature. When the reaction is substantially complete, typically after about 1 to about 10 hours, the mixture is poured into water and the aqueous phase is extracted with, for example, ethyl acetate. The desired compound is then isolated by solvent evaporation and crude purification can be accomplished by, for example, flash chromatography on silica gel eluting with ethyl acetate/hexane. Purification can be acomplished by, for example, flash chromatography on silica gel eluting with methylene chloride.
  • The compounds of formula 1 wherein L1 and L2 are both represented by carbonyl groups, L1 and L2 are both represented by sulfonyl groups or L2 is represented by a carbonyl group and L1 is represented by a sulfonyl group can be prepared by techniques and procedures well known an appreciated by one of ordinary skill in the art. A general synthetic scheme for preparing these compounds of formula 1 is set forth in Scheme C. In Scheme C, all substituents unless otherwise indicated are as previoulsy defined.
    Figure 00280001
  • Scheme C provides a general synthetic procedure for preparing the compounds of formula 1 wherein L1 and L2 are both represented by carbonyl groups, L1 and L2 are both represented by sulfonyl groups or L2 is represented by a carbonyl group and L1 is represented by a sulfonyl group.
  • In step a, the appropriate elastase inhibiting peptide fragment of formula 2 is coupled with the appropriate derivative of L as described by structure (17) to give the corresponding L-elastase inhibiting peptide fragment of structure (18) by techniques well known in the art.
  • When the compound of formula 1 is one wherein L1 and L2 are both represented by carbonyl groups, an appropriate derivative of L as described by structure (17) is one wherein the L2 carbonyl group is represented by a t-butyloxycarbonyl protected carboxylic acid and the L1 carbonyl group is represented by an unprotected carboxylic acid.
  • When the compound of formula 1 is one wherein L1 and L2 are both represented by sulfonyl groups, an appropriate derivative of L as described by structure (17) is one wherein the L1 sulfonyl group is represented by a sulfonyl chloride group and the L2 sulfonyl group is represented by an unprotected sulfonic acid.
  • When the compound of formula 1 is one wherein L2 is represented by a carbonyl group and L1 is represented by a sulfonyl group, an appropriate derivative of L as described by structure (17) is one wherein the L2 carbonyl group is represented by a t-butyloxycarbonyl protected carboxylic acid and the L1 sulfonyl group is represented by a sulfonyl chloride group.
  • In step b, the appropriate L-elastase inhibiting peptide fragment of structure (18) is coupled with the appropriate cathepsin G inhibiting peptide fragment of formula 3 to give the corresponding compound of formula 1 by techniques well known in the art.
  • When the appropriate L-elastase inhibiting peptide fragment of structure (18) is one wherein the L2 carbonyl group is represented by a t-butyloxycarbonyl protected carboxylic acid, the t-butyloxycarbonyl protected carboxylic acid must first be hydrolyzed by techniques well known in the art prior to the coupling reaction in step b.
  • Starting materials for use in Scheme C are readily available to one of ordinary skill in the art.
  • The compounds of formula 1 wherein L1 is represented by a carbonyl group and L2 is represented by a sulfonyl group can be prepared by techniques and procedures well known an appreciated by one of ordinary skill in the art. A general synthetic scheme for preparing these compounds of formula 1 is set forth in Scheme D. In Scheme D, all substituents unless otherwise indicated are as previously defined.
    Figure 00310001
  • Scheme D provides a general synthetic procedure for preparing the compounds of formula 1 wherein L1 is represented by a carbonyl group and L2 is represented by a sulfonyl group.
  • In step a, the appropriate cathepsin G inhibiting peptide fragment of formula 3 is coupled with the appropriate derivative of L as described by structure (19) to give the corresponding cathepsin G inhibiting peptide fragment of structure (20) by techniques well known in the art.
  • An appropriate derivative of L as described by structure (19) is one wherein the L1 carbonyl group is represented by a t-butyloxycarbonyl protected carboxylic acid and the L2 carbonyl group is represented by a sulfonyl chloride group.
  • In step b, the appropriate cathepsin G inhibiting peptide fragment of structure (20) is coupled with the appropriate elastase inhibiting peptide fragment of formula 2 to give the corresponding compound of formula 1 by techniques well known in the art.
  • The t-butyloxycarbonyl protected carboxylic acid functionality on L1 of the appropriate cathepsin G inhibiting peptide fragment of structure (20) must first be hydrolyzed by techniques well known in the art prior to the coupling reaction in step b.
  • Starting materials for use in Scheme D are readily available to one of ordinary skill in the art.
  • The following specific examples are given to illustrate the preparation of this invention although the scope of compounds is not meant to be limiting to the scope of compounds embraced by formula 1.
    • EXAMPLE 1 [CF3]Phe-Pro-Val-C(O)-phenylene-C(O)-Val-Pro-Val[CF2CF3]-- SEQ ID NO: 6 Preparation of Boc-Val[CF2CF3]
  • Dissolve Boc-Val dimethylhydroxyamide (1.0g, 3.8mmol) in ethyl ether (50mL) and cool to -78°C. Add pentafluoroethyl iodide (3g, 12.2mmol) followed by methyllithium•lithium bromide complex (6mL of a 1.5M solution). Repeat the addition of pentafluoroethyl iodide (3g, 12.2mmol) followed by methyllithium·lithium bromide complex (6mL of a 1.5M solution) three times. Stir for 15 minutes at -78°C then allow to warm to room temperature. Pour into water and separate the organic phase. Extract the aqueous phase with ethyl ether (3X150mL), combine the organic phases and dry (Na2SO4). Evaporate the solvent in vacuo and purify by silica gel chromatography (10% ethyl acetate/hexane) to give the title compound.
    • Preparation of Val[CF2CF3]•hydrochloride
  • Dissolve Boc-Val[CF2CF3] (350mg, 1.1mmol) in ethyl acetate (5OmL) and cool to 0°C. Treat with hydrogen chloride gas for 5 minutes and stir for 30 minutes. Remove the solvent invacuo to give the title compound.
    • Preparation of Boc-Val-Pro-Val[CF2CF3]
  • Dissolve Boc-Val-Pro (314mg, 1.0mmol) in methylene chloride (4mL) and add N-methylmorpholine (252mg, 2.5mmol). Cool to -22°C and add isobutylchloroformate (136mg, 1.0mmol). Stir for 20 minutes and add to Val[CF2CF3]•hydrochloride (1.1mmol). Stir for 1 hour at -22°C, allow to warm to room temperature and stir for 3 hours. Purify by silica gel chromatography (40% ethyl acetate/hexane) to give the title compound (405mg).
    • Preparation of Val-Pro-Val[CF2CF3]•hydrochloride
  • Dissolve Boc-Val-Pro-Val[CF2CF3] (385mg, 0.74mmol) in ethyl acetate (50mL) and cool to 0°C. Treat with hydrogen chloride gas for 5 minutes and stir for 30 minutes. Evaporate the solvent in vacuo to give the title compound (334mg).
  • Preparation of Boc-Val-Pro-Phe[OH][CF3]
  • Mix 2-phenyl-Δ2-oxazoline-4-phenylmethyl-5-one (Synthesis, #3, 191-3, (1982)) (300g) and trifluoroacetic anhydride (700g). Heat at reflux for 3 hours then stir overnight at room temperature. Evaporate the solvent in vacuo and add oxalic acid (400g). Stir and add additional oxalic acid (50g). Heat until evolution of CO2 ceases and a solid forms. Cool to room temperature and dissolve in a 1:3 mixture of water/ethyl acetate (12L). Separate the organic phase, wash until basic with saturated sodium hydrogen carbonate then with water. Dry (MgSO4), filter and concentrate by boiling to a volume of 2.5L. Cool to room temperature and add hexane (1L). Filter the precipitated solid and air dry to give 1,1,1-trifluoro-2-one-3-benzoylamino-4-phenylbutane (187.6g).
  • Dissolve 1,1,1-trifluoro-2-one-3-benzoylamino-4-phenylbutane (187.6g) in ethanol (1L) and cool in an ice bath. Add sodium borohydride (llg) in portions over 15 minutes. Remove the ice bath and stir at room temperature to 1.5 hours. Replace the ice bath and carefully treat with 10% hydrochloric acid (250mL). Add ethyl acetate (4L) to dissolve and then add water (500mL). Separate the organic phase, wash with brine (4X300mL) and dry (MgSO4). Filter and evaporate the solvent in vacuo. Add hexane and filter to give 1,1,1-trifluoro-3-benzoylamino-4-phenyl-2-butanol as a white solid (145.2g).
    • Mix 1,1,1-trifluoro-3-benzoylamino-4-phenyl-2-butanol (145.2g), concentrated hydrochloric acid (1.4L), water (700mL) and ethanol (1L). Heat to reflux for 24 hours then add additional concentrated hydrochloric acid (400mL) and ethanol (1.2L). Stir an additional 24 hours. Evaporate the ethanol in vacuo and filter. Cool the filtrate to room temperature and treat with sodium hydrogen carbonate and then with 50% sodium hydroxide while cooling in an ice bath. When pH 10 is obtained, filter off the solid to give [CF3][OH]-Phe (58.2g).
  • Dissolve Boc-Val-Pro (3.3g, 10.5mmol) in methylene chloride (25mL) and add N-methylmorpholine (2.12g, 21mmol). Cool to -22°C and add isobutylchloroformate (1.43g, 10.5mmol). Stir at -22°C for 25 minutes then add [CF3][OH]Phe (2.5g, 11.5mmol). Stir at -22°C for 3 hours, allow to warm to room temperature and stir overnight. Pour into water (100mL) and extract into ethyl ether (3X150mL). Wash the combined organic phases with dilute hydrochloride acid then saturated sodium hydrogen carbonate. Dry (Na2SO4) and evaporate the solvent in vacuo. Purify by silica gel chromatography (40% ethyl acetate/hexane) to give the title compound (5.27g).
    • Preparation of Boc-Val-Pro-Phe[CF3]
  • Dissolve Boc-Val-Pro-Phe[OH][CF3] (0.79g, 1.54mmol) in methylene chloride (25mL) and add Dess-Martin reagent (2.5g). Stir at room temperature overnight then pour into 50mL of water containing sodium hydrogen carbonate (1.0g) and sodium bisulfite (1.7g). Extract with ethyl ether (3X100mL) and dry (Na2SO4). Evaporate the solvent in vacuo and purify by silica gel chromatography (40% ethyl acetate/hexane) to give the title compound (755mg).
    • Preparation of Val-Pro-Phe[CF3]•hydrochloride
  • Dissolve Boc-Val-Pro-Phe[CF3] in ethyl acetate (100mL) and cool to 0°C. Treat with hydrogen chloride gas for 5 minutes and stir at 0°C for 30 minutes. Evaporate the solvent in vacuo to give the title compound (840mg).
    • Preparation of Boc-phenylene-C(O)-Val-Pro-Phe[CF3]
  • Dissolve Boc-phenylene-C(O)OH (370mg, 1.67mmol) in methylene chloride (4mL) and N-methylmorpholine (0.18mL, 1.67mmol). Cool to -20°C and add isobutylchloroformate (0.227g, 1.76mmol) and stir for 45 minutes. Add methylene chloride (2mL) and add N-methylmorpholine (0.18mL) and Val-Pro-Phe[CF3]•hydrochloride(0.75g, 1.67mmol). Stir at -20°C for 2 hours, allow to warm to room temperature and stir for an additional 3 hours. Pour into a mixture of methylene chloride (10mL) and water (20mL). Separate the organic phase and extract the aqueous phase with methylene chloride (2X20mL). Combine the organic phases and dry (Na2SO4). Evaporate the solvent in vacuo and purify by silica gel chromatography to give the title compound (575mg).
    • Preparation of H2OC-phenylene-C(O)-Val-Pro-Phe[CF3]•hydrochloride
  • Dissolve Boc-phenylene-C(O)-Val-Pro-Phe[CF3] (250mg) in ethyl acetate (50mL) and cool to 0°C. Treat with hydrogen chloride gas for 5 minutes and stir at 0°C for 1 hour. Evaporate the solvent in vacuo to give the title compound (232mg).
    • Preparation of [CF3]Phe-Pro-Val-C(O)-phenylene-C(O)-Val-Pro-Val[CF2CF3]--SEQ ID NO: 6
  • Dissolve H2OC-phenylene-C(O)-Val-Pro-Phe[CF3]•hydrochloride (230mg, 0.41mmol) in methylene chloride (3mL) and add N-methylmorpholine (41.4mg, 0.41mmol). Cool to -20°C and add isobutylchloroformate (55.7mg, 0.41mmol). Stir for 45 minutes at -20°C and add Val-Pro-Val[CF2CF3]•hydrochloride (185mg, 0.41mmol). Stir at -22°C for 3 hours, allow to warm to room temperature and stir for an additional 3 hours. Pour into water and extract into methylene chloride (3X25mL). Combine the organic phases and dry (Na2SO4). Evaporate the solvent in vacuo and purify by silica gel chromatography (ethyl acetate then methanol) to give the title compound (190mg).
    • Example 2 [CF3]Phe-C(O)-phenylene-C(O)-Val-Pro-Val[CF2CF3]--SEQ ID NO: 16 Preparation of Boc-phenylene-C(O)-Phe[OH][CF3]
  • Dissolve Boc-phenylene-C(O)OH (0.615g, 2.8mmol) in methylene chloride (6mL) and add N-methylmorpholine (0.6g). Cool to -22°C and add isobutylchloroformate (0.4mL, 3.08mmol). Stir at -22°C for 25 minutes and add a solution of Phe[OH][CF3] (0.64g, 2.9mmol) in methylene chloride (2mL) and N-methylmorpholine (0.3g). Stir at -22°C for 1 hour, allow to warm to room temperature and stir an additional 2 hours. Pour the mixture into water (100mL)and extract into ethyl ether (100mL then 50mL). Combine the organic phases and dry (Na2SO4). Evaporate the solvent in vacuo and purify by silica gel chromatography (40% ethyl acetate/hexane) to give the title compound (840mg).
    • Preparation of Boc-phenylene-C(O)-Phe[CF3]
  • Dissolve Boc-phenylene-C(O)-Phe[OH][CF3] (0.6g) in methylene chloride (25mL) and add Dess-Martin reagent (1.8g). Stir for 48 hour and pour into a mixture of sodium hydrogen carbonate (0.8g) and sodium bisulfite (1.41g) in water (25mL) and ethyl ether (100mL). Separate the organic phase and extract the aqueous phase with ethyl ether (50mL). Combine the organic phases and dry (MgSO4). Evaporate the solvent in vacuo and purify by silica gel chromatography (25% ethyl acetate/hexane) to give the title compound (430mg).
    • Preparation of H2OC-phenylene-C(O)-Phe[CF3]
  • Dissolve Boc-phenylene-C(O)-Phe[CF3] (216mg, 0.51mmol) in ethyl acetate (50mL) and cool to 0°C. Treat with hydrogen chloride gas for 5 minutes and stir for 3 hours. Evaporate the solvent in vacuo to give the title compound (197mg).
    • Preparation of [CF3]Phe-C(O)-phenylene-C(O)-Val-Pro-Val[CF2CF3]--SEQ ID NO: 16
  • Suspend H2OC-phenylene-C(O)-Phe[CF3] (175mg) in methylene chloride (2mL) and add N-methylmorpholine (100µL). Cool to -22°C and add isobutylchloroformate (65µL). Stir at -22°C for 25 minutes and add a solution of Val-Pro-Val[CF2CF3] (240mg) in methylene chloride (2mL) and N-methylmorpholine (60µL). Stir at -22°C for 30 minutes, allow to warm to room temperature and stir for an additional 1.5 hours. Evaporate the solvent in vacuo to approximately 1mL volume and purify by silica gel chromatography (50% ethyl acetate/hexane) to give the title compound (110mg).
    • Example 3 [CF3]Phe-Pro-Val-C(O)-Val-Pro-Val[CF2CF3]--SEQ ID NO: 17
  • Partition Val-Pro-Val[CF2CF3]•hydrochloride (200mg) between ethyl acetate (10mL) and saturated sodium hydrogen carbonate (20mL). Separate the organic phase and extract the aqueous phase with ethyl acetate (3X20mL). Combine the organic phases, dry (MgSO4) and evaporate the solvent in vacuo to give Val-Pro-Val[CF2CF3].
  • Partition Val-Pro-Phe[CF3]•hydrochloride (200mg) between ethyl acetate (10mL) and saturated sodium hydrogen carbonate (20mL). Separate the organic phase and extract the aqueous phase with ethyl acetate (3X20mL). Combine the organic phases, dry (MgSO4) and evaporate the solvent in vacuo to give Val-Pro-Phe[CF3].
  • Dissolve phosgene (545mg, 5.51mmol) in benzene (3.5mL) and slowly add a solution of Val-Pro-Val[CF2CF3] (568mg, 1.37mmol) in benzene (1mL). Stir at 60-75°C for several hours then reflux vigorously for 2 hours. Remove approximately 1/2 the benzene by distillation and cool the remaining solution in an ice bath. Add a solution of Val-Pro-Phe[CF3] (500mg, 1.37mmol) in ethyl ether (2mL) and stir at room temperature for several hours. Evaporate the solvent in vacuo and purify by chromatography to give the title compound.
    • Example 4 [CF3]Phe-Pro-Val-CH2-CH2-Val-Pro-Val[CF2CF3]--SEQ ID NO: 18
  • Dissolve Val-Pro-Val[CF2CF3] (568mg, 1.37mmol) and Val-Pro-Phe[CF3] (500mg, 1.37mmol) in methanol (20mL) and add glyoxal (200mg of a 40% solution in water, 1.37mmol), sodium cyanoborohydride (86mg, 1.37mmol) and 1 drop of 1% bromocresol green in ethanol. Maintain the pH of the reaction with 1N hydrochloric acid in methanol until the indicator no longer changes. Evaporate the solvent in vacuo and partition the residue between 1N sodium hydroxide (5mL) and ethyl acetate (10mL). Separate the organic phase, dry (MgSO4) and evaporate the solvent invacuo. Purify by chromatography to give the title compound.
    • SEQUENCE LISTING
  • (1) GENERAL INFORMATION:
  • (i) APPLICANT: Angelastro, Michael R
  • Bey, Philippe
  • Doherty, Niall S
  • Janusz, Michael J
  • Mehdi, Shujaath
  • Peet, Norton P
  • (ii) TITLE OF INVENTION: Inhibitors of Cathepsin G and Elastase for Preventing Connective Tissue Degradation
  • (iii) NUMBER OF SEQUENCES: 18
  • (iv) CORRESPONDENCE ADDRESS:
  • (A) ADDRESSEE: Marion Merrell Dov Inc.
  • (B) STREET: 2110 East Galbraith Rd.
  • (C) CITY: Cincinnati P. O. Box 156300
  • (D) STATE: Ohio
  • (E) COUNTRY: USA
  • (F) ZIP: 45215-6300
  • (v) COMPUTER READABLE FORM:
  • (A) MEDIUM TYPE: Floppy disk
  • (B) COMPUTER: IBM PC compatible
  • (C) OPERATING SYSTEM: PC-DOS/MS-DOS
  • (D) SOFTWARE: PatentIn Release #1.0, Version #1.25
  • (vi) CURRENT APPLICATION DATA:
  • (A) APPLICATION NUMBER: US 07/704,499
  • (B) FILING DATE: 23-MAY-1991
  • (C) CLASSIFICATION:
  • (viii) ATTORNEY/AGENT INFORMATION:
  • (A) NAME: Nesbitt, Stephen L
  • (B) REGISTRATION NUMBER: 28,981
  • (C) REFERENCE/DOCKET NUMBER: M01593
  • (ix) TELECOMMUNICATION INFORMATION:
  • (A) TELEPHONE: (513) 948-7965
  • (B) TELEFAX: (513) 948-7961
  • (C) TELEX: 214320
  • (2) INFORMATION FOR SEQ ID NO:1:
  • (i) SEQUENCE CHARACTERISTICS:
  • (A) LENGTH: 8 amino acids
  • (B) TYPE: amino acid
  • (D) TOPOLOGY: linear
  • (ii) MOLECULE TYPE: peptide
  • (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:
    Figure 00410001
  • (2) INFORMATION FOR SEQ ID NO:2:
  • (i) SEQUENCE CHARACTERISTICS:
  • (A) LENGTH: 4 amino acids
  • (B) TYPE: amino acid
  • (D) TOPOLOGY: linear
  • (ii) MOLECULE TYPE: peptide
  • (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:
    Figure 00410002
  • (2) INFORMATION FOR SEQ ID NO:3:
  • (i) SEQUENCE CHARACTERISTICS:
  • (A) LENGTH: 4 amino acids
  • (B) TYPE: amino acid
  • (D) TOPOLOGY: linear
  • (ii) MOLECULE TYPE: peptide
  • (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:
    Figure 00410003
  • (2) INFORMATION FOR SEQ ID NO:4:
  • (i) SEQUENCE CHARACTERISTICS:
  • (A) LENGTH: 4 amino acids
  • (B) TYPE: amino acid
  • (D) TOPOLOGY: linear
  • (ii) MOLECULE TYPE: peptide
  • (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:
    Figure 00420001
  • (2) INFORMATION FOR SEQ ID NO:5:
  • (i) SEQUENCE CHARACTERISTICS:
  • (A) LENGTH: 4 amino acids
  • (B) TYPE: amino acid
  • (D) TOPOLOGY: linear
  • (ii) MOLECULE TYPE: peptide
  • (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:
    Figure 00420002
  • (2) INFORMATION FOR SEQ ID NO:6:
  • (i) SEQUENCE CHARACTERISTICS:
  • (A) LENGTH: 6 amino acids
  • (B) TYPE: amino acid
  • (D) TOPOLOGY: linear
  • (ii) MOLECULE TYPE: peptide
  • (ix) FEATURE:
  • (A) NAME/KEY: Modified-site
  • (B) LOCATION: 1
  • (D) OTHER INFORMATION /note= "Xaa is a valine analog having a -CF2CF3 group replacing the hydroxy of the terminal carboxy."
  • (ix) FEATURE:
  • (A) NAME/KEY: Modified-site
  • (B) LOCATION: 3
  • (D) OTHER INFORMATION: /note= "Xaa is a valine analog having a -L1-Ph-L2- group replacing one of the hydrogens on the amino terminus wherein L1 and L2"
  • (ix) FEATURE:
  • (A) NAME/KEY: Modified-site
  • (B) LOCATION: 3
  • (D) OTHER INFORMATION: /note= "(cont) are each a carbonyl wherein L1 is bound to the peptide fragment containing the modified sites at locations 1-2 "
  • (ix) FEATURE:
  • (A) NAME/KEY: Modified-site
  • (B) LOCATION: 3
  • (D) OTHER INFORMATION: /note= "(cont)and L2 is bound to the peptide fragment containing the modified sites at locations 4-6 and Ph is a p-phenylene group."
  • (ix) FEATURE:
  • (A) NAME/KEY: Modified-site
  • (B) LOCATION: 6
  • (D) OTHER INFORMATION: /note= "Xaa is a phenylalanine analog having a -CF3 group replacing the hydroxy of the terminal carboxy"
  • (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:
    Figure 00430001
  • (2) INFORMATION FOR SEQ ID NO:7:
  • (i) SEQUENCE CHARACTERISTICS:
  • (A) LENGTH: 4 amino acids
  • (B) TYPE: amino acid
  • (D) TOPOLOGY: linear
  • (ii) MOLECULE TYPE: peptide
  • (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:
    Figure 00430002
  • (2) INFORMATION FOR SEQ ID NO:8:
  • (i) SEQUENCE CHARACTERISTICS:
  • (A) LENGTH: 4 amino acids
  • (B) TYPE: amino acid
  • (D) TOPOLOGY: linear
  • (ii) MOLECULE TYPE: peptide
  • (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:
    Figure 00440001
  • (2) INFORMATION FOR SEQ ID NO:9:
  • (i) SEQUENCE CHARACTERISTICS:
  • (A) LENGTH: 4 amino acids
  • (B) TYPE: amino acid
  • (D) TOPOLOGY: linear
  • (ii) MOLECULE TYPE: peptide
  • (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:
    Figure 00440002
  • (2) INFORMATION FOR SEQ ID NO:10:
  • (i) SEQUENCE CHARACTERISTICS:
  • (A) LENGTH: 4 amino acids
  • (B) TYPE: amino acid
  • (D) TOPOLOGY: linear
  • (ii) MOLECULE TYPE: peptide
  • (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:
    Figure 00440003
  • (2) INFORMATION FOR SEQ ID NO:11:
  • (i) SEQUENCE CHARACTERISTICS:
  • (A) LENGTH: 4 amino acids
  • (B) TYPE: amino acid
  • (D) TOPOLOGY: linear
  • (ii) MOLECULE TYPE: peptide
  • (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:
    Figure 00450001
  • (2) INFORMATION FOR SEQ ID NO:12:
  • (i) SEQUENCE CHARACTERISTICS:
  • (A) LENGTH: 4 amino acids
  • (B) TYPE: amino acid
  • (D) TOPOLOGY: linear
  • (ii) MOLECULE TYPE: peptide
  • (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:
    Figure 00450002
  • (2) INFORMATION FOR SEQ ID NO:13:
  • (i) SEQUENCE CHARACTERISTICS:
  • (A) LENGTH: 4 amino acids
  • (B) TYPE: amino acid
  • (D) TOPOLOGY: linear
  • (ii) MOLECULE TYPE: peptide
  • (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:
    Figure 00450003
  • (2) INFORMATION FOR SEQ ID NO:14:
  • (i) SEQUENCE CHARACTERISTICS:
  • (A) LENGTH: 4 amino acids
  • (B) TYPE: amino acid
  • (D) TOPOLOGY: linear
  • (ii) MOLECULE TYPE: peptide
  • (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:
    Figure 00460001
  • (2) INFORMATION FOR SEQ ID NO:15:
  • (i) SEQUENCE CHARACTERISTICS:
  • (A) LENGTH: 4 amino acids
  • (B) TYPE: amino acid
  • (D) TOPOLOGY: linear
  • (ii) MOLECULE TYPE: peptide
  • (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:
    Figure 00460002
  • (2) INFORMATION FOR SEQ ID NO:16:
  • (i) SEQUENCE CHARACTERISTICS:
  • (A) LENGTH: 4 amino acids
  • (B) TYPE: amino acid
  • (D) TOPOLOGY: linear
  • (ii) MOLECULE TYPE: peptide
  • (ix) FEATURE:
  • (A) NAME/KEY: Modified-site
  • (B) LOCATION: 1
  • (D) OTHER INFORMATION: /note= "Xaa is a valine analog having a -CF2CF3 group replacing the hydroxy of the terminal carboxy."
  • (ix) FEATURE:
  • (A) NAME/KEY: Modified-site
  • (B) LOCATION: 3
  • (D) OTHER INFORMATION: /note= "Xaa is a valine analog having a -L1-Ph-L2 group replacing one of the hydrogens on the amino terminus wherein L1 and L2
  • (ix) FEATURE:
  • (A) NAME/KEY: Modified-site
  • (B) LOCATION: 3
  • (D) OTHER INFORMATION: /note= "(cont) are each a carbonyl wherein L1 is bound to the peptide fragment containing the modified sites at locations 1-2 "
  • (ix) FEATURE:
  • (A) NAME/KEY: Modified-site
  • (B) LOCATION: 3
  • (D) OTHER INFORMATION: /note= "(coat) and L2 is bound to the peptide fragment containing the modified site at location 4 and Ph is a p-phenylene group."
  • (ix) FEATURE:
  • (A) NAME/KEY: Modified-site
  • (B) LOCATION: 4
  • (D) OTHER INFORMATION: /note= "Xaa is a phenylalanine analog having a -CF3 group replacing the hydroxy of the terminal carboxy."
  • (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:
    Figure 00470001
  • (2) INFORMATION FOR SEQ ID NO:17:
  • (i) SEQUENCE CHARACTERISTICS:
  • (A) LENGTH: 6 amino acids
  • (B) TYPE: amino acid
  • (D) TOPOLOGY: linear
  • (ii) MOLECULE TYPE: peptide
  • (ix) FEATURE:
  • (A) NAME/KEY: Modified-site
  • (B) LOCATION: 1
  • (D) OTHER INFORMATION: /note= "Xaa is a valine analog having a -CF2CF3 group replacing the hydroxy of the terminal carboxy."
  • (ix) FEATURE:
  • (A) NAME/KEY: Modified-site
  • (B) LOCATION: 3
  • (D) OTHER INFORMATION: /note= "Xaa is a valine analog having a -C(O)- group replacing one of the hydrogens on the amino terminus."
  • (ix) FEATURE:
  • (A) NAME/KEY: Modified-site
  • (B) LOCATION: 6
  • (D) OTHER INFORMATION: /note= "Xaa is a phenylalanine analog having the -CF3 group replacing the hydroxy of the terminal carboxy."
  • (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:
    Figure 00480001
  • (2) INFORMATION FOR SEQ ID NO:18:
  • (i) SEQUENCE CHARACTERISTICS:
  • (A) LENGTH: 6 amino acids
  • (B) TYPE: amino acid
  • (D) TOPOLOGY: linear
  • (ii) MOLECULE TYPE: peptide
  • (ix) FEATURE:
  • (A) NAME/KEY: Modified-site
  • (B) LOCATION: 1
  • (D) OTHER INFORMATION: /note= "Xaa is a valine analog having a -CF2CF3 group replacing the hydroxy of the terminal carboxy."
  • (ix) FEATURE:
  • (A) NAME/KEY: Modified-site
  • (B) LOCATION: 3
  • (D) OTHER INFORMATION: /note= "Xaa is a valine analog having a -(CH2)n+2- group wherein n is 0 replacing one of the hydrogens on the amino terminus.
  • (ix) FEATURE:
  • (A) NAME/KEY: Modified-site
  • (B) LOCATION: 6
  • (D) OTHER INFORMATION: /note= "Xaa is a phenylalanine analog having a -CF3 group replacing the hydroxy of the terminal carboxy."
  • (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:
    Figure 00480002

Claims (12)

  1. A process for preparing compound of the formula
    Figure 00540001
    wherein
    P1
    is Ala, bAla, Leu, Ile, Val, Nva, bval, Met, Nle, Gly, or Sar;
    P1'
    is Ala, bAla, Leu, Ile, Val, Nva, bVal, Met, Nle. Phe, Tyr, Tyr(Me), Ala(3pyr), Ala(4pyr), Trp, or Nal(1);
    P2
    is Pro, Ind, Ala, bAla, Leu, Ile, Val, Nva, bval, Met, Nle, Phe, Tyr, Tyr(Me), Ala(3pyr), Ala(4pyr), Trp, or Nal(1);
    P2'
    is Pro, Ind, Ala, bAla, Leu, Ile, Val, Nva, bval, Met, Nle, Gly, Sar or is absent;
    P3
    is Lys, Arg, Pro, Ind, Ala, bAla, Leu, Ile, Val, Nva, bVal, Met, or Nle;
    P3'
    is Ala, bAla, Leu, Ile, Val, Nva, bval, Met, Nle, Gly, Sar or is absent;
    P4
    is Ala, bAla, Leu, Ile, Val, Nva, bVal, Met, Nle, or is absent;
    P4'
    is Ala, bAla, Leu, Ile, Val, Nva, bval, Met, Nle, Gly, Sar, or is absent;
    L
    is a group of the formula
    Figure 00550001
    EIM and CGIM are each independently selected from the group consisting of -C(O)C(O)R, -CF2CF3, -CF3, CF2H, -CO2R3, -CONHR3, -CF2CHR3C(O)NHR, -H, alkyl, aryl, aralkyl, -C(O)R, wherein
    R3 is H, alkyl, phenyl, benzyl,
    R is OH or alkoxy
    or a pharmaceutically acceptable salt thereof, which comprises reacting of a compound of the formula
    Figure 00550002
    wherein P4, P3, P2, P1, L and EIM are as defined above, with a compound of the formula
    Figure 00560001
    wherein P4', P3', P2,' P1', and CGIM are as defined above, in the presence of a suitable coupling agent.
  2. A process for preparing a compound of claim 1 wherein CGIM and EIM are each independently either a -CF3 or -CF2CF3 group.
  3. A process for preparing a compound of any one of claims 1 or 2 wherein
    P1
    is norvaline or valine;
    P1'
    is phenylalanine;
    P2
    is proline;
    P2'
    is proline or is absent;
    P3
    is isoleucine, valine, or alanine;
    P3'
    is alanine, valine or is absent;
    P4
    is alanine or is absent; and
    P4'
    is alanine or is absent.
  4. A process for preparing a compound of any one of claims 1, 2 or 3 wherein -P4-P3-P2-P1- (SEQ ID NO: 2) is a -Ala-Ala-Pro-Val- (SEQ ID NO: 3); -Lys(2CBz)-Pro-Val-; or -Val-Pro-Val- group.
  5. A process for preparing a compound of any one of claims 1, 2, or 3 wherein -P4'-P3'-P2'-P1'- (SEQ ID NO: 4) is a -Ala-Ala-Pro-Phe- (SEQ ID NO: 5); Val-Pro-Phe-; or -Phe group.
  6. A process for preparing a compound of claim 1 which is
    Figure 00570001
EP92914209A 1991-05-23 1992-04-21 Inhibitors of cathepsin g and elastase for preventing connective tissue degradation Expired - Lifetime EP0587799B1 (en)

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US704499 1991-05-23
PCT/US1992/003288 WO1992020357A1 (en) 1991-05-23 1992-04-21 Inhibitors of cathepsin g and elastase for preventing connective tissue degradation

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PT100517B (en) 1999-06-30
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NZ242807A (en) 1994-07-26
HU217612B (en) 2000-03-28
EP0587799A4 (en) 1995-06-14
IL101925A0 (en) 1992-12-30
WO1992020357A1 (en) 1992-11-26
HUT65140A (en) 1994-04-28
ATE181925T1 (en) 1999-07-15
JPH06508355A (en) 1994-09-22
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AU2184192A (en) 1992-12-30
ZA923602B (en) 1993-02-24
US5510333A (en) 1996-04-23
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EP0587799A1 (en) 1994-03-23
NO934218L (en) 1993-11-22

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